A phage-encoded RNA-binding protein inhibits the antiviral activity of a toxin–antitoxin system

Abstract Bacteria harbor diverse mechanisms to defend themselves against their viral predators, bacteriophages. In response, phages can evolve counter-defense systems, most of which are poorly understood. In T4-like phages, the gene tifA prevents bacterial defense by the type III toxin–antitoxin (TA) system toxIN, but the mechanism by which TifA inhibits ToxIN remains unclear. Here, we show that TifA directly binds both the endoribonuclease ToxN and RNA, leading to the formation of a high molecular weight ribonucleoprotein complex in which ToxN is inhibited. The RNA binding activity of TifA is necessary for its interaction with and inhibition of ToxN. Thus, we propose that TifA inhibits ToxN during phage infection by trapping ToxN on cellular RNA, particularly the abundant 16S rRNA, thereby preventing cleavage of phage transcripts. Taken together, our results reveal a novel mechanism underlying inhibition of a phage-defensive RNase toxin by a small, phage-encoded protein.


Supplemental Figure and Table Legends
(D) Overlay of the crystal structure of P. atrosepticum ToxN (ToxNPa) and the predicted structure of E. coli ToxN (ToxNEc) in complex with TifARB69.
(E) Space-filling AlphaFold model for the ToxN-TifARB69 complex, with TifA residues predicted to interact with ToxN indicated.
(F) Per-residue confidence of five AlphaFold predictions for the structure of ToxN in complex with TifAT4.The top-ranked prediction was used for downstream analyses.
(G) Per-residue confidence of five AlphaFold predictions for the structure of ToxN in complex with TifARB69.The top-ranked prediction was used for downstream analyses.
(H) Heat map showing predicted aligned error (PAE) for five AlphaFold-predicted models for the ToxN-TifAT4 complex; a low PAE value indicates that AlphaFold predicts well-defined relative positions and orientations for the two domains or proteins.The highest-ranking model was used for analysis in the manuscript.
(I) Heat map showing predicted aligned error (PAE) for five AlphaFold-predicted models for the ToxN-TifARB69 complex.The highest-ranking model was used for analysis in the manuscript.

Table S1. Reagents, Assays, and Software
Critical commercial reagents, assays, and software used in this study.
Table S2.Protein sequences for ToxN, TenpN, and TifA homologs Protein sequences for ToxN, TenpN, and TifA homologs used for the neutralization screen in Figure 1.

Table. S3. Strains
E. coli strains generated and used in this study.

Table S4. Plasmids
Plasmids generated and used in this study.

Table S5. Primers and Oligos
DNA and RNA oligos used in this study.

Table S6. G-blocks
G-block sequences used to clone ToxN and TifA homologs used for the neutralization screen in

Bacterial and Virus Strains
For a complete list of bacterial strains used in this study, see Table S3.

RNA-Sequencing This study GSE234211
Recombinant DNA For a complete list of plasmids and G-blocks generated for this study, see Table S4.

Oligonucleotides
For a complete list of oligonucleotides used for this study, see Table S5.

Figure S1 .
Figure S1.TifA and ToxN homologs are present in T4-like phage and bacterial genomes, respectively

Figure S2 .
Figure S2.Rescue of ToxN homolog overexpression by co-expression of various TifA homologs

Figure S4 .
Figure S4.Use of a tab screen to isolate TifA-resistant ToxN mutants (B) Growth curves for MG1655 cells harboring pBR322-toxIN (wild type or mutant), with calculated doubling times indicated in brackets.

Figure S5 .
Figure S5.ToxN forms a high molecular weight RNA-protein complex when it is co-purified with TifA

Figure S6 .
Figure S6.TifA has a conserved nucleic acid-binding domain